Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties

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Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties. / Belenkaya, S. V.; Bondar, A. A.; Kurgina, T. A. et al.

In: Biochemistry (Moscow), Vol. 85, No. 7, 01.07.2020, p. 781-791.

Research output: Contribution to journal › Article › peer-review

BibTeX

@article{e7e7c6b2ec7342efb2c5de21a7bd837b,

title = "Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties",

abstract = "For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.",

keywords = "Michaelis–Menten kinetics parameters, milk-clotting activity, recombinant chymosin, thermal stability, CAMEL CHYMOSIN, PASTORIS, BOVINE CHYMOSIN, Michaelis-Menten kinetics parameters, ENZYME, CLONING, PURIFICATION, PREPROCHYMOSIN, ACTIVE CHYMOSIN",

author = "Belenkaya, {S. V.} and Bondar, {A. A.} and Kurgina, {T. A.} and Elchaninov, {V. V.} and Bakulina, {A. Yu} and Rukhlova, {E. A.} and Lavrik, {O. I.} and Ilyichev, {A. A.} and Shcherbakov, {D. N.}",

year = "2020",

month = jul,

day = "1",

doi = "10.1134/S0006297920070068",

language = "English",

volume = "85",

pages = "781--791",

journal = "Biochemistry (Moscow)",

issn = "0006-2979",

publisher = "Maik Nauka-Interperiodica Publishing",

number = "7",

}

RIS

TY - JOUR

T1 - Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties

AU - Belenkaya, S. V.

AU - Bondar, A. A.

AU - Kurgina, T. A.

AU - Elchaninov, V. V.

AU - Bakulina, A. Yu

AU - Rukhlova, E. A.

AU - Lavrik, O. I.

AU - Ilyichev, A. A.

AU - Shcherbakov, D. N.

PY - 2020/7/1

Y1 - 2020/7/1

N2 - For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.

AB - For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.

KW - Michaelis–Menten kinetics parameters

KW - milk-clotting activity

KW - recombinant chymosin

KW - thermal stability

KW - CAMEL CHYMOSIN

KW - PASTORIS

KW - BOVINE CHYMOSIN

KW - Michaelis-Menten kinetics parameters

KW - ENZYME

KW - CLONING

KW - PURIFICATION

KW - PREPROCHYMOSIN

KW - ACTIVE CHYMOSIN

UR - http://www.scopus.com/inward/record.url?scp=85087873890&partnerID=8YFLogxK

U2 - 10.1134/S0006297920070068

DO - 10.1134/S0006297920070068

M3 - Article

C2 - 33040722

AN - SCOPUS:85087873890

VL - 85

SP - 781

EP - 791

JO - Biochemistry (Moscow)

JF - Biochemistry (Moscow)

SN - 0006-2979

IS - 7

ER -

ID: 24752889